1. Field of the Invention
This invention relates to systems and methods for two-hybrid assays, in particular two-hybrid assays for identifying agents which interfere with protein-protein interactions. The invention further relates to systems and methods for identifying agents which interfere with protein-DNA interactions.
Specific protein-protein interactions are central to biological functions. Protein-protein interactions are intermolecular associations which occur when the surfaces of two or more proteins are precisely matched and a large number of non-covalent bonds form between them. Protein-protein interactions are involved for example in antibody-antigen reactions and in the interaction of cell surface receptors with specific ligands. Clearly the identification of inhibitors of specific protein-protein interactions will be useful for example in developing pharmaceuticals.
2. Description of Related Art
Investigation of protein-protein interactions under physiological conditions has been problematic. One approach has been the so-called "two-hybrid" system of Fields & Song (1) described in U.S. Pat. No. 5,283,173. The two-hybrid system involves the use of two chimeric genes which encode hybrid proteins to test for an interaction between a known protein and protein of interest in vivo. The first chimeric gene codes for a known protein, often called the bait protein, fused to the DNA-binding domain of a transcriptional activator. The second chimeric gene codes for a protein of interest fused to the transcriptional activation domain. Additionally, the protein of interest may not be known and could be derived for example from a cDNA library. In a suitable host cell such as yeast, if the protein of interest and the bait protein do interact they bring into proximity the DNA-binding and transcriptional activation domains. This proximity is sufficient to cause transcription of a marker gene placed under the control of a promoter containing a binding site for the DNA-binding domain.
The two-hybrid system generally allows detection of an interaction between two proteins by means of the positive signal of expression of a reporter gene. In the case of an assay to identify an agent which interferes with a protein-protein interaction, a negative signal indicating inhibition of the interaction might also be produced if the agent being tested is toxic to the cell. Thus, it would be preferable to use a system which gives a positive signal when the protein-protein interaction is inhibited.
WO 95/26400 describes a method for identifying inhibitors of protein-protein interactions using a reverse two-hybrid system, in which the protein-protein interaction drives the expression of a relay gene, which encodes a protein that represses the transcription and thus expression of a reporter gene. This provides indirectly a positive transcriptional read-out for molecules that inhibit the two-hybrid protein-protein interaction.